1. Field of the Invention
The invention relates to a Streptococcus suis adhesin protein, biologically or immunologically active derivatives and fragments thereof, a method for producing the adhesin protein, and an antibody raised against the adhesin protein, or the derivative or fragment thereof. The invention also relates to the use of pigeon ovomucoid or a synthetic derivative in the identification of the adhesin protein. The adhesin protein according to the invention, the derivatives and fragments thereof and their antibodies can be used both diagnostically, therapeutically, and prophylactically.
S. suis bacteria (the Lancefield group D) are known to cause meningitis, pneumonia, arthritis and sepsis in pigs. S. suis type 1 causes mainly septicemia and meningitis in newborn pigs, while type 2 causes meningitis in pigs about 3 to 10 weeks in age, and may also be infectious to humans. There are at least 29 capsular types identified, many of which cause disease in pigs. Concentration of pig breeding and increasing animal densities facilitate the spreading of infections, and infectious diseases caused by S. suis become more common. For this reason, the development of a diagnostic technique and a vaccine is of vital importance in the identification and prevention of meningitis and other serious infections. Selective preventive measures directed to pig breeders may also be possible.
2. Prior art
The first event in the establishment of an infectious disease is the adhesion of bacteria to the surface of host cells (Beachey, E. H. (1981) J. Infect. Dis. 143, 325-345). The adhesion of bacteria is often mediated by an adhesin protein which occurs on the bacterial surface and adheres specifically to cell surface receptor structures. As a consequence, adhesin is well-suited for the development of a vaccine as the antibodies are directed specifically against a factor necessary for the bacterium; in addition, the specificity allows detrimental side effects to be avoided.
A wide variety of adhesins are known, but in many cases their exact structure and mechanism still remain unknown. They are proteins that recognize the receptors of the host cell specifically, the receptors being usually carbohydrate structures. Various bacterial adhesins and carbohydrate structures recognized by them are described e.g. in Sharon, N., (1987) FEBS Letters, 217, 145-157.
E. coli and many other gram negative bacteria adhere to specific molecules on the surface of the host cell by lectin-like bacterial adhesins. Usual adhesin receptors include the sugar components of glycolipids and glycoproteins. Adhesins are often attached to hair-like structures called fimbriae (pili) on the surface of the bacterial cell. There are many different types of fimbriae; they vary with respect to both structure and sugar specificity.
As there are often bacterial receptor structures on erythrocytes, bacteria adhere to these structures and agglutinate erythrocytes in vitro. Bacterial cultures may express three or four hemagglutinins (adhesins), each having a different binding specificity. Accordingly, the bacterium is capable of adhering to various cell types.
In enterobacteria studies, adhesion reactions are divided into two main classes: mannose sensitive (MS) reactions in which the hemagglutination reaction is inhibited by .alpha.-mannosides, and mannose resistant (MR) reactions in which the hemagglutination cannot be inhibited by .alpha.-mannosides. E. coli type 1 fimbriae (MS structure) consists almost solely of identical 17 kDa subunits. Several MR adhesins recognize an .alpha.-Gal(1-4)-.beta.-Gal structure (P-specific adhesin) or an .alpha.-NeuNAc-(2-3)-.beta.-Gal structure (S-specific adhesin). In general, purified fimbriae consist of subunits having a molecular weight varying between 15 and 22 kDa.
Adhesin protein is usually a distinct protein attached to the tips or sides of the fimbriae; it may also be attached directly to the outer membrane of the bacterium. In certain bacterial strains the surface of the bacterial cell contains adhesin protein even in the absence of fimbriae. In such cases, adhesins may form an adhesin capsule around the bacterium. The size of the non-fimbrial adhesins of E. coli varies between 13 and 28 kDa (Jann, K. and Hoschutzky, H. (1990), Current Topics in Microbiology and Immunology 151, 55-70).
It is known that streptococci bind to various soluble proteins and glycoproteins, whereas their oligosaccharide specificity is mostly unknown. Their specific binding to epithelial cells is also nearly unknown. Certain Streptococcus sanguis strains recognize galactose and sialic acid containing structures (Murray, P. A., Levine, M. J., Tabak, L. A., and Reddy, M. S. (1982) Biochem. Biophys. Res. Commun. 106, 390-396). The binding of Streptococcus pneumoniae to epithelial cells is inhibited by GlcNAc.beta.1-3Gal (Andersson, B., Porras, O., Hanson, L. A., Lagergard, T., and Svanborg-Eden, C. (1986) J. Infect. Dis. 153, 232-237), and this bacterium has been reported to bind to GalNAc.beta.1-4Gal-containing glycolipids in the lung (Krivan, H. C., Roberts, D. D. and Ginsburg, V. (1988) Proc. Natl. Acad. Sci., USA, 85, 6157-6161).
S. mitis adheres to the surface of a tooth and contributes to the formation of dental plaque. A sialic-acid-binding adhesin has been successfully isolated from this bacterial strain. The sialic-acid-binding protein had at least two disulphide-bound subunits of 96 kDa and 70 kDa. Both subunits bound N-acetylneuraminic acid-.alpha.2-3-galactose-.beta.1-3-N-acetylgalactosamine (Murray, P. A., Levine, M. J., Reddy, M. S., Tabak, L. A., Bergey, E. J. (1986) Infect. Immun. 53, 359-365). The galactose-binding adhesin of S. sanguis was determined to have a molecular weight of about 20 kDa, and its isoelectric point was in the range 8.5-9 (Nagata, K., Nakao, M., Shibata S., Shizu-kuishi, S., Nakamura R. and Tsunemitsu, A. (1983) J. Periodontol. 54, 163-172). In addition to this, a 36-kDa adhesin protein has been cloned from S. sanguis bacteria. The carbohydratic specificity of the cloned adhesin remains unknown, whereas it is known that adhesin adheres to saliva-coated hydroxyapatite through the mediation of a pH sensitive receptor (Ganeskumar, N., Song, M. and McBride, B. C. (1988) Infect. Immun. 56, 1150-1157).
S. suis is an important pathogen in pigs. It colonizes the tonsils or nostrils of piglets and causes serious infections. There are several capsular serotypes, for which reason no vaccine effective against all S. suis types is available. The capsular polysaccharides and surface proteins of S. suis have been reported to play a role in the pathogenesis, but the molecular mechanism of the infection has remained unknown. Two strains of S. suis bacteria have been shown to bind to sialylated poly-N-acetyllactosamine glycans (Liukkonen J., Haataja, S., Tikkanen, K., Kelm, S., and Finne, J. (1992) J. Biol. Chem. 267, 21105-21111). However, the hemagglutination caused by most S. suis bacteria is inhibited by galactose, so that the galactose-recognizing adhesin is probably more common in S. suis bacteria than that mentioned above (Kurl, D., Haataja, S. and Finne, J. (1989) Infect. Immun. 57, 384-389).
Several attempts have been made to detach and isolate adhesins by heat treatment and/or extraction. The adhesin of Proteus mirabilis was isolated by heat treatment (65.degree. C., 20 min, 50 mM sodium phosphate (pH 7.2) with 2 M urea), and purified by gel filtration (Sepharose CL-4B) (Wray, S. K., Hull, S. I., Cook, R. G., Barrish, J. and Hull, R. A. (1986) Infect Immun. 54, 43-49). The lectin of S. mitis was separated by extracting the bacteria with lithium-3,5-diiodosalicylate, and a sialic-acid-binding protein was purified from the extract by gel filtration and affinity chromatography (Murray, P. A., Levine, M. J., Reddy, M. S., Tabak, L. A. and Bergey, E. J. (1986) Infect. Immun. 53, 359-365). A protein of Streptococcus pyogenes (the Lancefield group A) capable of adhering to the cardiac tissue and the basement membrane of kidney cells was successfully separated by treating the bacteria with alkali for 18 h (Stinson, M. W. and Bergey E. J. (1982) Infect. Immun. 35, 335-342). A non-fimbrial adhesin of E. coli was extracted by heating the bacterial suspension at 65.degree. C. for 30 min (Goldhar, J., Perry, R., Golecki, J. R., Hoschutzky, H., Jann B., and Jann K. (1987) Infect. Immun., 55, 1837-1842). Fimbriae were separated from the fimbrial E. coli by mechanical homogenization, and the adhesin protein was separated from the fimbrial homogenate by heat treatment (70.degree. C., 1 h, PBS/5 mM EDTA) (Moch, T., Hoschutzky, H., Hacker, J., Kroncke, K.-D. and Jann, K. (1987) Proc. Natl. Acad. Sci. USA 84, 3462-3466) After detachment the adhesin protein has often been precipitated by ammonium sulphate precipitation, whereafter the adhesins have been purified further by various methods. Conventional methods used for the separation of adhesins, however, have not been suitable for the isolation of S. suis adhesins.